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目的:建立一种高效扩增人T细胞受体(TCR)β链可变区(Vβ)基因的方法。方法:根据TCRVβ26个亚家族基因序列特点设计扩增Vβ基因的上游内引物34条、上游外引物37条,并将内、外上游引物各分为8个简并组。在恒定区(C区)设计下游内、外和测序引物各1条以及扩增Cβ基因的上游引物1条。提取细胞RNA,用PolyA介导反转录后,采用巢式PCR扩增正常人CD8T细胞TCRVβ26个亚家族基因,并用JurkatT淋巴瘤细胞作为对照。用T-easy载体克隆RT-PCR产物,对克隆基因进行测序。结果:从正常人的CD8T细胞中扩增到所有TCRVβ亚家族基因,克隆后测序与相应的参考序列同源。从Jurkat细胞扩增出TCRVβ8基因,经测序验证与文献报道一致,且最少可从10个细胞提取的RNA模板中扩增成功。结论:建立的巢式RT-PCR方法可以高效、广谱地扩增人TCRVβ基因,为进一步进行抗原特异性细胞毒T淋巴细胞的TCR克隆和功能研究打下了良好基础。
Objective: To establish a method for efficiently amplifying the β chain variable region (Vβ) gene of human T cell receptor (TCR). Methods: According to the characteristics of TCRVβ26 subfamily genes, 34 upstream primers and 37 upstream primers were designed and synthesized. The inner and outer primers were divided into 8 degenerate groups. In the constant region (C region), one downstream internal and external sequencing primer and one upstream primer to amplify Cβ gene were designed. Cell RNA was extracted and PolyA-mediated reverse transcription was used to amplify TCRVβ26 subfamily genes of normal human CD8T cells by nested PCR. JurkatT lymphoma cells were used as control. The RT-PCR product was cloned using the T-easy vector and the cloned gene was sequenced. Results: All TCRVβ subfamily genes were amplified from normal human CD8T cells. The cloned sequences were homologous to the corresponding reference sequences. The TCRVβ8 gene was amplified from Jurkat cells and verified by sequencing, which was consistent with the reported results and was successfully amplified from RNA templates extracted from 10 cells. CONCLUSION: The established nested RT-PCR method can amplify human TCRVβ gene efficiently and broadly, which lays a good foundation for further TCR cloning and function study of antigen-specific cytotoxic T lymphocytes.